Abstract

The development of economical and high-throughput gene synthesis technology has been
hampered by the high occurrence of errors in the synthesized products, which requires
expensive labor and time to correct. Here, we describe an error correction reaction
(ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors
from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing
of the synthetic gene products. Mismatches are recognized and excised by a combination
of mismatch-specific endonuclease and 3'→5' exonuclease activities in the reaction
mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles
the resulting fragments into intact genes. The process can be iterated for increased
fidelity. With two iterations, we were able to reduce errors in synthetic genes by
>16-fold, yielding a final error rate of ∼1 in 8700 bp.